1. Field of the Invention
The present disclosure relates to pro-carboxypeptidase B and carboxypeptidase B and to a process for preparing them.
2. Discussion of the Background Art
Carboxypeptidase B (CPB) is a pancreatic exopeptidase which cleaves by the hydrolysis of peptide linkages at basic amino acids, such as lysine, arginine and ornithine. The cleavage is effected at the C-terminal end of the polypeptides. It is a zinc-containing peptidase (EC 3.4.17.2).
Carboxypeptidase B is formed from pre-pro-carboxypeptidase B, which is enzymatically inactive. From pre-pro-carboxypeptidase B, a signal peptide is cleaved off to obtain a pro-carboxypeptidase B, which is also enzymatically inactive. From the latter, another peptide is cleaved off to obtain the active carboxypeptidase.
The molecular weight of carboxypeptidase B is about 35 kD. It is employed for a wide variety of purposes, especially for the preparation of peptides, such as insulin, and in protein sequence analysis. Carboxypeptidase B is usually purified from porcine pancreas.
The cDNA sequences of human carboxypeptidase B are known.
WO 96/23064 describes a process for the preparation of recombinant rat carboxypeptidase B. Attempts to express the plasmid described were not successful.
Commercially available carboxypeptidase (purified from natural sources) typically has activities of about 50 to 170 U/mg. One unit (1 U) corresponds to the hydrolysis of 1 mmol of hippuryl-L-Arg per min at 25° C. and at a pH of 7.65.
Carboxypeptidase B purified from natural sources is always contaminated with small amounts of other proteases. Therefore, there is still a need for highly pure carboxypeptidases having an activity as high as possible.